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In this study, characterization of cellular mechanics of W303 yeast cells was conducted using nanoneedles inside an environmental SEM (ESEM). This enhanced ESEM system comprises a standard ESEM instrument as a nanoimaging tool, a cooling stage as a humidity controller for cellular biology and 7 degrees of freedom and linear actuators as nanomanipulator/effector. Four types of nanoneedles were used in our experiments, i.e., silicon (Si), titanium (Ti) coated Si, and two types of tungsten (W) nanoneedles. The Si and Ti nanoneedles were fabricated using 2 N/m spring constant cantilevers. While the W nanoneedles were fabricated using 0.09 and 2 N/m spring constant cantilevers (W0.09 and W2 nanoneedles). The Si, silicon-titanium (Si-Ti), W0.09, and W2 nanoneedles are suitable to be used for local stiffness characterization of single cells. This capability can be used in future for fast disease detection since disease cells may show different cell mechanics properties as compared to the normal cells. The Si-Ti and W2 nanoneedles can penetrate the cell without cell bursting, and this could be important in single cell surgery in future to avoid cell damage.